Reverse transcription is the process by which a retrovirus such as HIV-1 converts its single-stranded (ss) RNA genome into a double-stranded (ds) DNA copy that is integrated into host chromosomal DNA. This process is catalyzed by the virion-associated enzyme, reverse transcriptase (RT). However, another viral protein, the nucleocapsid protein (NC), is also required for productive viral DNA synthesis. (A) We study the mechanistic basis for NC activity. HIV-1 NC is a small (7 kDa), basic nucleic acid binding protein with two zinc fingers (ZFs), each containing the invariant CCHC Zn-coordinating motifs. It is also a nucleic acid chaperone, i.e., NC facilitates remodeling of nucleic acid structures so that the most thermodynamically stable conformations are formed. This property is critical for promoting efficient and specific reverse transcription. (i) Recent studies have focused on Gag C-terminal cleavage products i.e., NCp15 (NCp9-p6), NCp9 (NCp7-SP2), and mature NC (NCp7), and comparison of their nucleic acid chaperone activities in reconstituted systems modeling early reverse transcription events, i.e., annealing and elongation steps in tRNALys3-primed (-) strong-stop DNA synthesis and subsequent minus-strand transfer. The maximum levels of annealing are similar for all of the proteins, but there are important differences in their ability to facilitate RT-catalyzed DNA extension. Thus, at low concentrations, NCp9 has the greatest activity, but with increasing concentrations, DNA synthesis is significantly reduced. This finding reflects NCp9s strong aggregating activity and nucleic acid binding affinity (associated with the highly basic C-terminal SP2 domain), which together with its slow dissociation kinetics, limit the ability of RT to traverse the nucleic acid template during DNA elongation. NCp15 has much lower minus-strand transfer activity than NCp9 or NCp7, which we attribute to NCp15's acidic C-terminal p6 domain. Indeed, mutants with Ala substitutions in the acidic residues have improved chaperone activity. Viewed collectively, our findings help to explain why complete processing of the NC precursors is critical for long-term virus replication and fitness. (ii) In view of the importance of monkeys for evaluating candidate AIDS vaccines, it is of interest to analyze relationships between simian and human viral systems. Since reverse transcription is a target for anti-HIV strategies, we initiated comparative studies of the simian immunodeficiency virus (SIV) and HIV-1 NCs. In studies of NC activity in reconstituted systems modeling the reactions involved in minus-strand transfer, our results indicate that HIV-1 activity is about 2- to 3-fold higher than the activity of SIV NC. Moreover, both NCs show very similar qualitative interactions with DNA in single molecule DNA stretching experiments (performed by our collaborators), reflecting similarity of the domain structure of the two proteins. However, in the quantitative analysis, there is a significant difference in the compaction force generated at extensions less than the DNA contour length. This may be due to the shorter N-terminal domain of SIV NC relative to HIV-1 NC and the higher charge density in the ZF domains of the HIV-1 protein. In turn, this would likely contribute to the stronger aggregation activity of HIV-1 NC (an important component of nucleic acid chaperone activity), consistent with the minus-strand transfer results. Additional collaborative studies are now in progress to evaluate the relative contribution of specific vs. non-specific NC-RNA binding using salt titration analysis and small angle X-ray scattering (SAXS). (B) Our interest in host proteins that affect HIV-1 reverse transcription and replication has led us to investigate the activities of human APOBEC3 (A3) proteins. (i) We have been studying A3A, which like other A3 family members, is a cytidine deaminase that converts dC residues to dU in ssDNA and functions as a DNA mutator. In fact, A3A signature mutations have recently been found to be associated with a variety of tumors. A3A also inhibits a wide range of viruses, including retroviruses, and displays potent activity against endogenous retroelements such as LINE-1. Our collaborators have determined the solution structure of A3A at high resolution using NMR spectroscopy. We have now performed structure-guided mutagenesis studies designed to characterize A3A's enzymatic, nucleic acid binding, and biological activities. Surprisingly, although A3A also binds ssRNA, NMR analysis demonstrates that the RNA and DNA binding interfaces differ. Moreover, no deamination of ssRNA is detected in real-time NMR assays. In experiments on LINE-1 retrotransposition, assays with active- and non-active site A3A mutants reveal that the absence of deaminase activity per se does not always result in loss of anti-LINE-1 activity, demonstrating that these two activities are not linked. We have also performed experiments that indicate a mechanism for A3As ability to mutate normally ds genomic DNA, an activity that is implicated in carcinogenesis. Taken together, our studies provide new insights into the molecular properties of A3A and its role in multiple cellular and antiviral functions. (ii) In earlier work (Iwatani et al. 2007), we found that A3G inhibits RT-catalyzed HIV-1 DNA elongation reactions in a deaminase-independent manner due to its strong nucleic acid binding affinity and slow dissociation kinetics. We proposed that the inability of RT to traverse the RNA or DNA template creates a roadblock (hence the name roadblock mechanism). Recent single-molecule DNA stretching experiments provide support for this mechanism and show that A3G initially binds ssDNA with rapid on-off rates (compatible with enzymatic activity) and then subsequently converts to a slowly dissociating nucleic acid binding protein as A3G slowly oligomerizes on the viral genome, thereby inhibiting reverse transcription. In contrast, an A3G oligomerization-deficient mutant does not exhibit the slow off rate, but is still catalytically active. This result demonstrates that the roadblock activity of A3G is regulated via protein oligomerization, which may be a general property of other A3 proteins that exhibit deaminase-independent activity. The paper describing this work (1) was the subject of a News and Views commentary (Nat Chem (2014) 6:13-14). (iii) Other work centered on human A3H, which has seven haplotypes. Hap II restricts HIV-1 and occurs with high frequency in Africa. We have identified the determinants of human A3H Hap II cytidine deaminase and anti-HIV-1 activities, using site-directed sequence- and structure-guided mutagenesis. We constructed a homology model of A3H based on the known structure of the A3G C-terminal domain. The model reveals a large cluster of basic residues that are likely to be involved in nucleic acid binding. Not surprisingly, mutation of these residues to acidic or neutral amino acids results in reduction or in most cases, abrogation of enzymatic activity. Mutations in a structural element that dictates substrate specificity also negatively impact catalytic function. Interestingly, as shown for A3G, some of the A3H mutants that are defective in catalysis (including the catalytic mutant E56A) are able to restrict HIV-1 replication, although at a lower efficiency than wild type. This result raises the possibility that A3H inhibits HIV-1 by deaminase-dependent and -independent mechanisms. Endogenous RT assays with E56A mutant virions showing reduced synthesis of viral DNA and the fact that A3H can multimerize strongly suggest that a roadblock mechanism might also be relevant to A3H deaminase-independent HIV-1 restriction. Taken together, our work should contribute to continuing efforts to combat HIV infection in the African human population.